Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of presumed autoimmune etiology. MS is characterized by focal lesions (plaques) in the brain and spinal cord leading to progressive neurological dysfunction. The etiology of MS is unknown, but it is thought to result from a combination of genetic and environmental factors. Currently, there is no specific test for diagnosing MS and the diagnosis relies on recognition of the clinical history of the subject. The diagnosis can be supported by MRI of the brain and spinal cord, analysis of the cerebrospinal fluid, and evoked potential studies of the visual and somatosensory pathways. In addition, systemic or infectious etiologies with similar presentation must be excluded. Multiple sclerosis may progress and regress unpredictably; however, there are several patterns of symptoms. Approximately 85-90% of patients begin with a relapsing-remitting (RRMS) course and 40% eventually become progressive (secondary progressive MS, SPMS); in 10%, MS presents a primary progressive course (PPMS). The different MS subtypes are characterized by the past course of the disease (e.g. unpredictable relapses, remissions and progression of neurologic decline). From a clinical perspective, patients with different disease courses show different treatment responses. For instance, patients with relapsing-remitting MS are more likely to respond to immunomodulatory therapy than those with a progressive disease course (Bitsch and Bruck, CNS Drugs, 2002; 16(6):405-18). Thus, characterizing the MS subtype is important not only for prognosis but also for therapeutic decisions.
MS is not only heterogeneous in its clinical symptoms and rate of progression, but also in its response to therapy and histopathological findings (Lucchinetti et al., 2000, Ann Neurol 47, 707-17). The pattern of active demyelination is identical among multiple lesions examined from a given MS patient, yet heterogeneous between patients, suggesting pathogenic heterogeneity. Pattern I is characterized by T-cell/macrophage-mediated demyelination. Pattern II is characterized by antibody/complement-associated demyelination. Pattern III is defined by a distal oligodendrogliopathy, and pattern IV is characterized by oligodendrocyte degeneration in the periplaque white matter; to date pattern IV has only been identified in autopsy cases. Patterns I and II lesions show the typical perivenous distribution and sharp borders that are the pathological hallmarks of MS lesions and are thought to result from classical autoimmune mechanisms (Lucchinetti, et al., 2004, Ann Neurol 56, 308). MRI is commonly used to visualize MS lesions in vivo. The use of MRI to study MS lesions is limited, however, because it cannot provide information about the pathological composition of the lesions. From a clinical standpoint patients with Pattern II, but not Pattern I, have been reported to respond to plasmapheresis (Keegan et al., 2005, Lancet 366, 579-82). Thus, there is a need for identifying patients that would be responsive to treatment with plasmapheresis.
The McDonald criteria was introduced in 2001, and revised in 2005 (Polman et al., 2006, Ann Neurol.; 59(4):727-8), as guidelines to facilitate early and accurate diagnosis of multiple sclerosis (MS). Diagnostic classifications are reduced to a) having MS, b) not having MS, or c) having possible MS. Advantages to the Criteria include the capability of making a definitive diagnosis of MS either after a monosymptomatic presentation or in the context of a primary progressive course. However, the diagnostic classification scheme and MRI criteria remain complicated and tedious, and this complexity limits their use in everyday practice. Furthermore, the specificity of these criteria is relatively low, emphasizing the importance of clinical judgment in excluding other diagnoses. In addition, studies have observed that standard MS disease-modifying medications can benefit patients who do not yet fulfill these diagnostic criteria. Finally, the McDonald criteria decreased the time required for MS diagnosis substantially, however it is still limited for those individuals who are diagnosed with possible MS, or those who will eventually receive a diagnosis of PPMS.
Although MS is considered a T cell mediated disease, several pieces of evidence support a role for B cells in the disease (Archelos et al., 2000, Ann Neurol. 47, 694-706). B cells can contribute to MS progression by their secretion of antibodies and cytokines, or by acting as antigen presenting cells (APC) to activate pathogenic T cells. B cells are significantly more efficient in processing and presenting antigens recognized by the antibodies they produce. Thus, it is not surprising that linear B and T cell epitopes are co-localized in CNS antigens targeted by the autoimmune response in human and experimental models of MS (Meinl et al., 2006, Ann Neurol. 59, 880-92; Wucherpfennig et al., 1997, J Clin Invest. 100, 1114-22).
Immune System Biomarkers
The immune system in both its innate and adaptive arms can be viewed as a type of biological health-maintenance system. In physiological terms, the cells and molecules comprising the immune system are considered to act to manage inflammation (Cohen, 2000, Academic Press, London). Inflammation is classically defined as the collective processes activated by injury that lead to healing. The immune system, by the way it initiates and manages inflammation, maintains the body by healing wounds, containing pathogens, organizing the structure of connective tissue, growing (angiogenesis) or destroying blood vessels, triggering regeneration of certain organs, activating the apoptosis of aged cells and those with irreparable DNA damage, degrading accumulations of abnormal molecules, disposing of waste, and performing other vital activities (Cohen, 2000). These varied expressions of inflammation maintain the integrity of the organism in response to its relentless post-developmental decomposition due to neoplasia, environmental injuries and infections, accumulations of metabolic products, waste, and other intoxications, and the inexorable advance of entropy.
The possibility that cerebrospinal fluid (CSF) antibodies in MS patients are generated as a response to myelin self-antigens has been investigated in detail. Antibodies reactive with several CNS antigens have been described, including those directed against myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-specific protein (OSP), myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein, 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase) and ab-crystallin. When analyzed, the subclasses of these antibodies correlated with a pro-inflammatory immune response. Many of these autoantibodies have been detected also in blood (Lalive et al., 2006, Proc Natl Acad Sci US. 103, 2280-5). In addition, higher titers of antibodies reactive with non myelin autoantigens (Annunziata et al., 1999, J Neurol Sci. 168, 32-6; Barned et al., 1995, Neurology. 45, 384-5; Colaco et al., 1987, Clin Exp Immunol. 68, 313-9; Roussel et al., 2000, J Autoimmun. 14, 259-65; Spadaro et al., 1999, Mult Scler. 5, 121-5) and to pathogens (Cepok et al., 2005, J Clin Invest. 115, 1352-60) have been also found in MS patients.
The role played by antibodies in MS still awaits further clarification. Antibodies to conformational epitopes in MOG have been purified from MS lesions and shown to alter the physiology of CNS cells (Lalive et al., 2006). Accordingly, U.S. Pat. App. Pub. No. 2005/0009096 provides methods utilizing detection or quantification of autoantibodies to specific epitopes of myelin/MOG components for diagnosis or prognosis of MS.
Antibodies reactive with linear epitopes in CNS antigens have also been isolated from MS lesions (Dalakas, 2006, Pharmacol Ther. 112, 57-70; Genain et al., 1999, Nat. Med. 5, 170-5), suggesting that they also play a direct role in MS pathology. Moreover, antibodies to MBP isolated from MS patients have been shown to have direct proteolytic activity (Ponmarenko et al., 2006, Proc Natl Acad Sci US. 103, 281-6). U.S. Pat. App. Pub. No. 2003/0092089 relates to an assay for detecting MBP autoantibodies, and alternatively in conjunction with the measurement of other biochemical markers associated with MS and related diseases.
Biomarkers are anatomic, physiologic, biochemical or molecular parameters associated to specific disease states. The search for MS biomarkers has been focused on indicators of the general activity of the inflammatory process. Several biomarkers aim at following not the inflammatory process itself, but its consequences such us neurodegeneration and axonal loss. Thus, altered levels of neurofilament light chains, tau and 14-3-3 protein have been described to correlate with axonal loss in MS patients.
Since MS is felt to be an organ specific autoimmune disorder, immune biomarkers have the potential to reflect disease activity and its response to therapy. Several large-scale proteomic studies have attempted the characterization of antibodies in CSF and serum, aiming to identify yet unknown targets of the autoimmune attack in MS patients (Lefran et al., 2004, J Immunol 172, 669-78). Moreover, specific antibodies have been investigated as biomarkers in MS, resulting in the identification of several up-regulated antibody responses to myelin antigens in CSF and/or serum. However, these biomarkers were not generalizable to the majority of MS patients or could not be validated in independent studies (Rinaldi and Gallo, 2005, Neurol Sci. 26, S215-7; Lim et al., 2005, Mult Scler. 11, 492-4). Similarly to what has been observed in other autoimmune diseases such as diabetes (Quintana et al., 2004, Proc Natl Acad Sci, 14615-21) and systemic lupus erythematosus (Li et al., 2005, J Clin Invest. 115, 3428-39), it is possible that no single biomarker will be conclusive, but rather a pattern of several biomarkers forming a fingerprint will be required.
The Antigen Chip
Antigen microarrays are newly developed tools for the high-throughput characterization of the immune response (Robinson et al., 2002, Nat Med 8, 295-301), and have been used to analyze immune responses in vaccination and in autoimmune disorders (Robinson et al., 2002; Robinson et al., 2003, Nat. Biotechnol. 21, 1033-9; Quintana et al., 2004; Kanter et al., 2006, Nat Med 12, 138-43). It has been hypothesized, that patterns of multiple reactivities may be more revealing than single antigen-antibody relationships (Quintana et al., 2006, Lupus 15, 428-30) as shown in previous analyses of autoimmune repertoires of mice (Quintana et al., 2004; Quintana et al., 2001, J Autoimmun 17, 191-7) and humans (Merbl et al., 2007, J Clin Invest 117, 712-8; Quintana et al., 2003, J Autoimmun 21, 65-75) in health and disease. Thus, autoantibody repertoires have the potential to provide both new insights into the pathogenesis of the disease and to serve as immune biomarkers (Cohen, 2007, Nat Rev Immunol. 7, 569-74) of the disease process.
Antigen microarrays have been used to characterize serum autoantibodies in systemic lupus erythematosus, rheumatoid arthritis and neuromyelitis optica. However, high-affinity specific antibodies in MS have not been reported with any regularity in serum (Meinl et al., 2006, Ann Neurol. 59, 880-92; O'Connor et al., 2007, Nat Med 12, 12; Zhou et al., 2006, Proc Natl Acad Sci US. 103, 19057-62). In contrast to autoantibodies in serum, Kanter and associates have used microarrays to detect lipid (Kanter et al., 2006) and αB-crystallin (Ousman et al., 2007, Nature. 448, 474-9) reactive antibodies in the CSF. Strikingly, the antibodies to αB-crystallin were of low affinity, detectable at 1:20 dilution (Ousman et al., 2007).
PCT Pub. No. WO 02/08755 to some of the inventors of the present invention is directed to a method, system and an article of manufacture for clustering and thereby identifying predefined antigens reactive with undetermined immunoglobulins of sera derived from patient subjects in need of diagnosis of disease or monitoring of treatment. The '755 publication discloses the use of antigen arrays for identifying antigens reactive with immunoglobulins of sera derived from subjects afflicted with various diseases. Further disclosed are diagnostic methods, and systems useful in these methods, employing the step of clustering a subset of antigens of a plurality of antigens, said subset of antigens being reactive with a plurality of antibodies being derived from a plurality of patients having an impaired immune system and suffering from a disease, and associating or deassociating the antibodies of a subject with the resulting cluster. While WO 02/08755 discloses methods useful in diagnosis of MS among other autoimmune diseases, there is no disclosure of diagnosing different subtypes of MS or monitoring MS progression.
U.S. Pat. App. Pub. No. 2005/0260770 to some of the inventors of the present invention discloses an antigen array system and diagnostic uses thereof. The application provides a method of diagnosing an immune disease, and particularly type 1 diabetes, or a predisposition thereto in a subject, comprising determining a capacity of immunoglobulins of the subject to specifically bind each antigen probe of an antigen probe set. The antigen probe set comprises a plurality of antigen probes selected from the group consisting of at least a portion of a cell/tissue structure molecule, at least a portion of a heat shock protein, at least a portion of an immune system molecule, at least a portion of a homopolymeric polypeptide, at least a portion of a hormone, at least a portion of a metabolic enzyme, at least a portion of a microbial antigen, at least a portion of a molluscan antigen, at least a portion of a nucleic acid, at least a portion of a plant antigen, at least a portion of a plasma molecule, and at least a portion of a tissue antigen, wherein the binding capacity of the immunoglobulin of the subject is indicative of the immune disease or the predisposition thereto. However, none of the prior art discloses an antigen array that can provide a specific, reliable, accurate and discriminatory assay for diagnosing MS, specifically for discriminating between different subtypes of MS and predicting or monitoring disease progression. Such discriminatory assays would be highly valuable in tailoring adequate therapeutic approach for each patient.
PCT Pub. No. WO 07/137,410 relates to methods for the diagnosis MS, different forms of MS or another demyelinating disorder. Particularly, WO 07/137,410 relates to specific metabolites, identified by their molecular masses, found to have different abundances or intensities between clinically diagnosed MS or other neurological disorders, and normal patients. Nevertheless, WO 07/137,410 does not disclose nor mention the use of testing an antibody reactivity pattern for identifying unique signature patterns in different subtypes of MS, and to further differentiate between patients having MS and those afflicted with other neurological disorders.
Thus, there remains a need for improved diagnostic methods and kits useful in diagnosing MS and particularly, diagnosing subtypes of MS in a subject.